title: Small molecule injection into single-cell C. elegans embryos via carbon-reinforced nanopipettes.
abstract: The introduction of chemical inhibitors into living cells at specific times in development is a useful method for investigating the roles of specific proteins or cytoskeletal components in developmental processes. Some embryos, such as those of Caenorhabditis elegans, however, possess a tough eggshell that makes introducing drugs and other molecules into embryonic cells challenging. We have developed a procedure using carbon-reinforced nanopipettes (CRNPs) to deliver molecules into C. elegans embryos with high temporal control. The use of CRNPs allows for cellular manipulation to occur just subsequent to meiosis II with minimal damage to the embryo. We have used our technique to replicate classical experiments using latrunculin A to inhibit microfilaments and assess its effects on early polarity establishment. Our injections of latrunculin A confirm the necessity of microfilaments in establishing anterior-posterior polarity at this early stage, even when microtubules remain intact. Further, we find that latrunculin A treatment does not prevent association of PAR-2 or PAR-6 with the cell cortex. Our experiments demonstrate the application of carbon-reinforced nanopipettes to the study of one temporally-confined developmental event. The use of CRNPs to introduce molecules into the embryo should be applicable to investigations at later developmental stages as well as other cells with tough outer coverings.
authors.affiliation: Department of Physics - Laboratory of Atomic and Solid State Physics, Cornell University, Ithaca, New York, United States of America.
authors.forename: Lucy D
authors.initials: LD
authors.lastname: Brennan
authors.forename: Thibault
authors.initials: T
authors.lastname: Roland
authors.forename: Diane G
authors.initials: DG
authors.lastname: Morton
authors.forename: Shanna M
authors.initials: SM
authors.lastname: Fellman
authors.forename: SueYeon
authors.initials: S
authors.lastname: Chung
authors.forename: Mohammad
authors.initials: M
authors.lastname: Soltani
authors.forename: Joshua W
authors.initials: JW
authors.lastname: Kevek
authors.forename: Paul M
authors.initials: PM
authors.lastname: McEuen
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: Kemphues
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Wang
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000008:administration & dosage; D017173:Caenorhabditis elegans / Q000187:drug effects; D029742:Caenorhabditis elegans Proteins / Q000378:metabolism; D002244:Carbon / Q000008:administration & dosage*; D016764:Cell Polarity / Q000187:drug effects; D016503:Drug Delivery Systems / Q000295:instrumentation / Q000379:methods; D004625:Embryo, Nonmammalian / Q000187:drug effects*; D007267:Injections / Q000295:instrumentation*; D008540:Meiosis / Q000187:drug effects; D054852:Small Molecule Libraries / Q000008:administration & dosage*; D053778:Thiazolidines / Q000008:administration & dosage
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D029742:Caenorhabditis elegans Proteins; D054852:Small Molecule Libraries; D053778:Thiazolidines; C087977:par-2 protein, C elegans; C512642:par-6 protein, C elegans; D002244:Carbon; C037067:latrunculin A